Recent discoveries about small non-coding RNAs (sncRNAs) have significantly advanced human genetics and molecular biology, largely due to the identification of a fundamental role of microRNAs (miRNAs), the best- characterized sncRNA family, as gene regulators. tRNA-derived RNA Fragments (tRFs) are a recently discovered sncRNA family that is ubiquitously expressed in organisms ranging from prokaryotes to humans, yet their biological functions and the mechanism(s) underlying those functions are largely unknown. Studies by our group have shown that the sncRNAs most highly induced by respiratory syncytial virus (RSV) belong to the tRF family, while the change in miRNA expression is minimal. These induced tRFs are functional and involved in the regulation of RSV replication. Notably, at least three tested tRFs have a gene trans- silencing function that is mechanistically distinct from that of miRNAs. However, more experimental evidence is needed to determine whether there is a widespread impact of mammalian tRFs on the regulation of gene expression, and more importantly, what mechanism(s) tRFs use for their gene regulatory function. In this project we propose the novel hypothesis that functional tRFs induced by RSV have a common gene trans-silencing function. Since RSV-induced tRFs are localized in the cytoplasm (and so are unlikely to play a role in regulating gene transcription, usually a nuclear event), we also hypothesize that the gene-suppression effect of tRFs occurs at post-transcriptional steps, e.g., mRNA stability or translation. In this exploratory project, these hypotheses will be tested in two Specific Aims. In Aim 1, tRFs important for RSV replication, viral gene expression and associated chemokine/cytokine induction will be identified. We will then determine whether these functional tRFs have gene trans-silencing activity. Aim 2 will test our second hypothesis that the gene- suppression effect of tRFs occurs at post-transcriptional steps. High-throughput methods, including microarray or proteomics analyses, will then be used to identify target candidates for a representative tRF. Target candidates will be experimentally confirmed by qRT-PCR and Western blot analysis. A mutagenesis study will also be performed to confirm that the effect of the tRF on its target(s) is direct and specific. This project may hav important translational implications by suggesting new therapeutic opportunities to modulate viral replication, providing experimental evidence to build a database of functional tRFs, and of more importance to identify the consensus features of tRF*target interaction for the development of a tRF target- prediction algorithm, which will undoubtedly facilitate the discovery of new gene regulatory networks and so will broadly benefit the research community. These goals will be explored in a future R01 application based on the results of this exploratory project.